Some scientific and commercial applications require the continuous delivery of a secondary liquid into a flow stream of a primary liquid, where the liquid mix ratios and flow rates must remain constant. This task can be unreliable at high liquid pressures (such as between 200-10,000 psi) where liquid compressibility becomes a factor, and/or where pumping pressures might fluctuate by several percent (such as between piston pump strokes). The task can be even more challenging when the secondary to primary liquid mix ratios are highly diluted (such between 1:100 and 1:10,000).
High performance liquid chromatography (HPLC), widely used to detect and identify different components of a test sample, is commonly operated under these demanding conditions. A typical HPLC instrument is schematically illustrated in FIG. 1, including a reciprocating pump 1 serially connected by capillary lines 2, 4, 6, 8 through sampler 3, separation column 5 and detector 7, to empty into waste container 9. A computer 11 controls the operation of the HPLC instrument and can display and retain test results.
The separation column 5 is filled with a selective stationary phase (such as of powder absorbents) to provide a high mobile phase flow resistance and different mobility rates of the sample components to be analyzed. Containers 13, connected by lines 14 through a degasser 12 and a proportional valve 15 to the inlet of pump 1, hold premixed liquid solvent and buffer combinations selected for yielding specific acidic or basic properties and/or ion strengths etc. needed to react properly with the test sample.
Typical solvents might include water, methanol, acetonitrile, hexane; while typical buffers might include phosphoric acid, trifluoroacetic acid (TFA), hydrochloric acid (HCl), triethyl amine (TEA), sulfuric acid, sodium phosphate, acetic acid-triethyl amine mixture, sodium alkyl sulfate, etc.
Operation of the HPLC instrument provides that buffered solvent(s) from the containers are continuously forced by pump 1 under high pressures (up to 5,000 psi.) to flow at a constant rate (such as 100-3,000 microliters per minute) through the serially arranged downstream lines and components 2-8 and into waste container 9. Periodically, a small quantity of test sample (a few microliters) will be injected at sampler 3 into this flowing buffered solvent stream. When the sample, moving somewhat as an isolated slug within the stream, reaches the separation column 5, its components will penetrate through the stationary phase at different rates for isolated detection and identification in the detector 7.
As the buffer/solvent ratio generally is highly diluted, possibly between 1:100 and 1:10,000, each HPLC test might require very few microliters of a concentrated buffer.
The inventor has recognized significant drawbacks to premixing the buffer and solvent, storing the mixture in containers 13, and passing such buffered mixture internally through the HPLC components including the degasser 12, the proportional valve 15, pump 1, and sampler 3.
For example, the buffered solvent can crystallize on these surfaces after extended instrument nonuse or down time. The buffered solvent can also be a good media for microorganism growth and/or contamination can build up on all wetted surfaces within these components. While flushing the flow passages with solvent between consecutive tests removes most such contaminates, lingering traces cumulatively might produce background noises that hinder detector sensitivity and/or reliability.
Also, the buffered solvent generally must be manually and tediously premixed, involving weighing and/or proportioning the different solvents, buffers or other chemical ingredients needed to achieve a desired pH, etc. This procedure can be expensive considering the time and chemical costs. Further, as a buffered solvent frequently is prepared in multi-liter quantities, if it should prove to be inappropriate for a proposed test method (and is of no immediate use otherwise), it might only be disposed of as waste. The limited storage life of a buffered solvent might even dictate that it be freshly prepare immediately before an intended HPLC test.
Further complicating manual preparation of the buffered solvent, some buffers, such as trifluoroacetic acid (TFA) and triethylamine (TEA), are hazardous and require a fume hood in order to be handled. Also, corrosive buffers, such as hydrochloric acid (HCl), can be routed internally only through special HPLC instruments that cost about twice as much as a regular HPLC instrument.